Protein Globularization During Folding. A Study by Synchrotron Small-angle X-ray Scattering

Semisotnov, Gennady V.; Kihara, Hiroshi; Kotova, Nina V.; Kimura, Kazumoto; Amemiya, Yoshiyuki; Wakabayashi, Katsuzo; Serdyuk, Igor N.; Timchenko, A. A.; Chiba, Kaori; Nikaido, Kiyokazu; Ikura, Teikichi; Kuwajima, Kunihiro

doi:10.1006/jmbi.1996.0535

Cited by 172 publications

(152 citation statements)

References 46 publications

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“…Of these, the prosthetic group containing cytochrome c and five highly charged, intrinsically unfolded proteins (unfolded in water) were excluded from our data set (see below). For the few proteins for which multiple denatured-state R G determinations have been reported by SAXS, we adopted data by using the following criteria (in order of preference): data collected over a range of denaturant concentrations (see Table 1) under a single set of conditions and fitted to determine the average unfolded R G value (27) or data collected at the highest GuHCl or urea concentration reported to date (28).…”

Section: N-tert-butyl-␣-(4-pyridyl)nitrone Nј-oxide Immediately Beformentioning

confidence: 99%

Random-coil behavior and the dimensions of chemically unfolded proteins

Kohn

Millett

Jacob

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

661

758

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Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (RG) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The RG of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r2 = 0.988) by a power-law relationship with a best-fit exponent, 0.598 ± 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble

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Section: N-tert-butyl-␣-(4-pyridyl)nitrone Nј-oxide Immediately Beformentioning

confidence: 99%

Random-coil behavior and the dimensions of chemically unfolded proteins

Kohn

Millett

Jacob

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

661

758

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“…(2) Associating intermediate states; recent work supports that equilibrium intermediate states in the thermal or solventinduced unfolding of proteins may show a strong tendency to associate (Filimonov & Rogov, 1996;Semitsonov et al, 1996). Associated intermediates, however, are unlikely to be significantly populated in the urea-induced unfolding of HEW lysozyme at pH 2.9, since their presence would have caused a protein concentration dependence of the denaturation profiles; this dependence would have been easily revealed by our data, which span a 200-fold change in protein concentration.…”

Section: Experimental Data Reported In This Work Do Not Provide Suppomentioning

confidence: 99%

Are There Equilibrium Intermediate States in the Urea-Induced Unfolding of Hen Egg-White Lysozyme?

Ibarra‐Molero

Sanchez-Ruiz

1997

Biochemistry

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Protein folding intermediates that are sometimes populated at equilibrium under mild denaturing conditions have attracted much attention as plausible models for the kinetic intermediates transiently populated in the refolding kinetic pathways. Hen egg-white lysozyme is often considered as a typical example of close adherence to the equilibrium, two-state unfolding mechanism. However, recent smallangle X-ray scattering studies suggest that an equilibrium intermediate state is significantly populated in the urea-induced unfolding of this protein at moderately acidic pH. In this work, we analyze the ureainduced unfolding of hen egg-white lysozyme on the basis of steady-state fluorescence measurements, characterization of the folding-unfolding kinetics, double-jump unfolding assays for the amount of native protein, and double-jump refolding assays for the amount of unfolded protein. Our results do not provide support for the presence of an intermediate state and, in particular, disfavor that the following two types of intermediates be significantly populated at equilibrium: (1) intermediates showing a substantial quenching of the tryptophan fluorescence (such as that observed in the transient intermediates found in the refolding kinetic pathway under strongly native conditions) and (2) associating intermediates. Also, the deconvolution of the radius of gyration unfolding profile by using the values for the amount of native state derived from our double-jump unfolding assays is consistent with a two-state unfolding equilibrium and suggests, furthermore, that, in this case, large alterations in the average structure of the unfolded ensemble do not take place in response to changes in urea concentration. This work points up possible pitfalls in the experimental detection of equilibrium folding intermediates and suggests procedures to circumvent them.

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“…1). Moreover, this discrepancy seems to be nearly universal among single-domain proteins: whereas the results of at least a half-dozen SAXS studies on chemically unmodified, single-domain proteins fail to detect any experimentally significant evidence (at experimental precision of, typically, a few percent) of contraction (8)(9)(10)(11)(12)(13)(14)(15), all of the more than one-dozen smFRET studies of dye-labeled, singledomain proteins reported to date have been interpreted in terms of unfolded states that contract as the denaturant concentration is lowered to ∼1 M (2,6,7,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

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confidence: 99%

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

Watkins

Simon

Sosnick

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond-or hydrophobically driven contraction of the unfolded state at low denaturant.protein folding | SAXS | two state | statistical coil | Flory scaling R ecent years have seen a significant controversy arise regarding the behavior of the unfolded states of single-domain proteins in response to changing levels of chemical denaturant. That is, although small-angle X-ray scattering (SAXS) and single-molecule FRET (smFRET) results are all consistent with the argument that, at high levels of chemical denaturant (∼6 M or above), unfolded proteins adopt a swollen, self-avoiding ensemble well-approximated as an excluded volume random coil (1-3), the two approaches produce seemingly discrepant results for the dimensions of the unfolded states populated at lower (∼1 M) denaturant (4). For example, time-resolved SAXS studies of the unfolded state transiently populated when protein L is rapidly shifted from high guanidine hydrochloride (GuHCl) to low denaturant conditions produce no experimentally significant evidence for the compaction of this single-domain protein before folding (4, 5) [e.g., estimated radii of gyration of 25.1 ± 0.3 Å for the unfolded state at equilibrium in 7 M GuHCl and 24.9 ± 1.1 Å for the transient unfolded state populated at 0.67 M GuHCl; confidence intervals are SEs (4)]. In contrast, multiple smFRET studies conducted at equilibrium suggest that the unfolded state of dye-labeled protein L contracts by 20-40% over this same range of denaturant concentrations (6, 7) (Fig.

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Protein Globularization During Folding. A Study by Synchrotron Small-angle X-ray Scattering

Cited by 172 publications

References 46 publications

Random-coil behavior and the dimensions of chemically unfolded proteins

Random-coil behavior and the dimensions of chemically unfolded proteins

Are There Equilibrium Intermediate States in the Urea-Induced Unfolding of Hen Egg-White Lysozyme?

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

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